Aspergillus sydowii and its application in preventing and treating tomato leafminer and tomato spider mite

Abstract

The application relates to the technical field of microbial inoculants, in particular to Aspergillus tamarii and application thereof, that is, application of the Aspergillus tamarii in prevention and treatment of Tetranychus truncatus Ehara and tomato leaf roller. The application discloses Aspergillus tamarii XJ-2, which has a preservation number of CCTCC NO: M 2023661; and a nucleotide sequence of SEQ ID NO. 1. The application also simultaneously provides the use of the above-mentioned Aspergillus tamarii XJ-2 in prevention and treatment of Tetranychus truncatus Ehara and tomato leaf roller, and the Aspergillus tamarii XJ-2 can also be used in prevention and treatment of Leptinotarsa decemlineata and caterpillar.

Description

Technical Field

[0001] The invention relates to the field of microbial inoculants, and in particular to a type of Aspergillus and its use in the control of truncate spider mite and tomato leafminer. Background Technology

[0002] Chemical control is currently one of the common control measures for Tetranychus carmine and the tomato leafminer. However, frequent and unscientific use of chemical agents has induced resistance in these mites and leafminers. The increasing resistance levels have led to increased application rates and frequencies of pesticides, which will further enhance the resistance levels of these mites and leafminers, increasing control costs and difficulties. Furthermore, the large-scale application of chemical pesticides also increases pesticide residues in agricultural products and the soil environment, causing adverse effects on human health and the ecological environment. Therefore, exploring integrated control methods with biological control as the main component has become a hot topic in the control of Tetranychus carmine.

[0003] In Xinjiang, the occurrence of the spider mite (hereinafter referred to as "spider mite") is widespread and severe. Spider mites are characterized by their small size, high reproductive rate, significant generation overlap, strong adaptability, and wide distribution. Nymphs and adults congregate on the leaves, tender stems, buds, and fruits of host plants, sucking sap, causing chlorosis, affecting photosynthesis, and ultimately leading to reduced or even complete crop yields, resulting in significant economic losses. Spider mites are an important target for the control of agricultural and forestry crops. Currently, spider mites commonly infest more than 100 economic crops, including those in the Poaceae (corn), Malvaceae (cotton), Leguminosae (soybeans, mung beans, red beans, kidney beans, etc.), Solanaceae (potatoes, eggplants, tomatoes, peppers, etc.), and Cucurbitaceae (cucumbers, pumpkins, watermelons, winter melons, etc.). Currently, spider mite control mainly relies on chemical pesticides. However, due to the extensive use of acaricides such as abamectin, pyridaben, fenpyroxime, and thiamethoxam, spider mites have developed varying degrees of resistance, making chemical control ineffective and rendering recommended dosages largely useless. In addition, the abuse and overuse of pesticides during chemical control can also kill non-target organisms, disrupt the ecological balance, and cause a resurgence of mites.

[0004] Tomato leafminer (Tuta absoluta (Meyrick)) is a newly invasive major vegetable pest in my country. Chemical control is currently one of the common control measures for tomato leafminer. However, due to the tomato leafminer's concealed nature, mainly burrowing into tomato leaves and fruits, its strong concealment often leads to poor control effects. Furthermore, frequent use of chemical agents has also induced the development of pesticide resistance in tomato leafminer. Currently, tomato leafminer has developed varying degrees of resistance to pyrethroids, abamectin, fenitrothion, permethrin, spinosad, etc. (Yang Shiyou, Zhang Rui, Li Honglin, Huang Jingmei, Kong Qiong, Yuan Shengyong. The effect of pesticide adjuvants on reducing the dosage and enhancing the efficacy of indoxacarb in controlling tomato leafminer [J / OL]. Journal of Environmental Entomology: 1-11 [2023-03-26]).

[0005] In summary, the small size, short generation time, rapid development, and high susceptibility to pesticide resistance of the truncated spider mite, coupled with the burrowing feeding behavior of the tomato leafminer larvae, necessitate increased application rates and frequencies of insecticides. This frequent application leads to a rapid increase in pesticide resistance. Clearly, controlling the truncated spider mite and the tomato leafminer is extremely difficult. Currently, both mites and leafminers are primarily controlled using chemical pesticides. However, the extensive use of acaricides such as abamectin, pyridaben, azoxystrobin, and thiamethoxam, as well as insecticides like pyrethroids and indoxacarb, has resulted in varying degrees of pesticide resistance in both mites and leafminers. Chemical control is often ineffective, and recommended dosages are largely useless. Furthermore, the overuse and misuse of pesticides during chemical control can also harm non-target organisms, disrupting the ecological balance and causing pest resurgence. Therefore, exploring efficient and environmentally friendly biological control resources is an urgent need in production. Fungal insecticides are characterized by their ability to infect through the body wall, their environmental friendliness, and their capacity for mass production, making them suitable for the control of this type of pest.

[0006] Utilizing entomopathogenic fungi is a key measure in the biological control of agricultural pests, known as "fungal control of insects," and a crucial means of reducing pesticide use and controlling pests. Beyond their environmental friendliness, broad pathogenicity against economically important pests, and safety for vertebrates, they also promote crop growth and increase yields. Therefore, exploring efficient and environmentally friendly biological control resources is an urgent need in production.

[0007] Biological control of pests is environmentally friendly and does not cause harm to humans and livestock. Its environmentally friendly characteristics can avoid a series of problems caused by chemical control. Currently, there are reports on the pathogenicity of microorganisms such as Bacillus thuringiensis, Beauveria bassiana, and Metarhizium anisopliae against Tetranychus truncatula and Tomato leafminer (Zhang Xiaona, Li Bin, Deng Jiao, et al. Research progress on biological control of Tetranychus truncatula [J]. Southern Agriculture, 2018, 12(02): 5-6; Liang Yongxuan, Guo Jianyang, Wang Qijing, et al. Research progress on biological control of Tomato leafminer. Acta Tropicalis, 2023, 14(1): 17-29).

[0008] The known uses of Aspergillus tamarii are:

[0009] 1. The invention CN115093983A, "A Growth-Promoting and Disease-Resistant Biological Agent", states that it has different degrees of promoting effect on the growth of Paris polyphylla seedlings and has an antagonistic effect on Phytophthora indicum.

[0010] 2. It has been reported that Aspergillus oryzae is highly toxic to melon flies (Yang Ye, Wang Meng, Ma Xiaoyan, Lu Binbing, Xu Zhanhua. Aspergillus fungi infecting melon flies and their biological characteristics [J]. Acta Mycologica Sinica, 2016, 35(01):20-28).

[0011] It should be noted that different strains have different toxicities to specific pests (Lorenz SC, Humbert P, Wassermann M, Mackenstedt U, Patel A VA broad approach to screening of Metarhizium spp. blastospores for the control of Ixodes ricinus nymphs[J]. Biological Control, 2020, 146(8):104270.).

[0012] Biological agents or formulations can reduce or replace the use of chemical pesticides, thereby achieving the goal of preventing and controlling spider mites while avoiding the threats to humans and the environment caused by chemical pesticides. A search revealed no reports on the use of Aspergillus to control truncate spider mites and tomato leafminers. Summary of the Invention

[0013] The technical problem to be solved by the present invention is to provide a *Aspergillus tamarii* XJ-2 and its uses.

[0014] To solve the above technical problems, the present invention provides Aspergillus tamarii XJ-2, whose accession number is CCTCC NO:M 2023661; its nucleotide sequence is as described in SEQ ID NO.1.

[0015] The present invention also provides the uses of the above-mentioned Aspergillus XJ-2: for the control of truncated spider mites and tomato leaf miners; and for the control of potato beetles and codling moths.

[0016] Note: The truncated spider mite and the tomato leafminer belong to the order Acari and order Lepidoptera pests, respectively.

[0017] This invention involves collecting diseased corn borer larvae from corn stalks in Zakuqi Niulu Village, Zakuqi Niulu Township, Chabuchar Xibe Autonomous County, Ili Kazakh Autonomous Prefecture, Xinjiang. Pure strains were isolated and purified in the laboratory, and identified as *Aspergillus tamarii* by morphology and molecular biology, with the strain named XJ-2. Toxicity activity tests of strain XJ-2 against four insect species showed high toxicity against *Tetranychus truncatula* and *Tomato Leafminer*, and some activity against *Potato Beetle* and *Potato Cactus*. The results are shown in Table 1. After inoculation into the body wall, dissection revealed no hyphae in healthy control insects, while a large number of vegetative hyphae were observed in the pathogenic insects. A brown mold layer was also found on the insects killed by *Aspergillus tamarii*. This indicates that *Aspergillus tamarii* XJ-2 primarily invades the insect body wall by producing appressorium and germ tubes. It then grows and reproduces rapidly inside the insect, damaging the insect's physiological functions and ultimately causing the insect's death.

[0018] This shows that Aspergillus has high insecticidal activity against both the truncated spider mite and the tomato leafminer, and it has great potential for biological control of pests. Attached Figure Description

[0019] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0020] Figure 1 To construct a phylogenetic tree of the target strain based on the ITS gene sequence (NJ method).

[0021] Figure 2 Morphological observation of the target strain (Aspergillus strain XJ-2);

[0022] Figure 2 In the middle: the top left is a front view of Aspergillus XJ-2 on a PDA, the top right is a back view of Aspergillus XJ-2 on a PDA, the bottom left is a conidia of Aspergillus XJ-2, and the bottom right is a conidiophore of Aspergillus XJ-2.

[0023] Figure 3 Image of a diseased Tetranychus truncatula infected with Aspergillus XJ-2.

[0024] Figure 4 The effect of Aspergillus XJ-2 on the hatching rate of tomato leafminer eggs.

[0025] Figure 5 The effect of Aspergillus XJ-2 on the mortality rate of tomato leafminer larvae at different instars.

[0026] Figure 6 Image of a 3rd instar tomato leafminer larva infected with Aspergillus XJ-2.

[0027] Figure 7 The effect of Aspergillus XJ-2 on the emergence rate of tomato leafminer moth.

[0028] Figure 8 Image of a diseased tomato leafminer pupa infected with Aspergillus XJ-2. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0030] Example 1: Isolation and Identification of Strains

[0031] Diseased corn borer larvae collected from corn plants in Zakuqi Niulu Village, Zakuqi Niulu Township, Chabuchar Xibe Autonomous County, Ili Kazakh Autonomous Prefecture, Xinjiang (longitude: 81.248489, latitude: 43.803871) were brought back to the laboratory. The dead corn borer larvae were sequentially soaked in 70% alcohol for 30 seconds, then in 0.1% mercuric chloride solution for 3 minutes (for thorough disinfection), and finally rinsed three times with sterile water. The treated corn borer larvae tissue was then inoculated onto PDA medium using an inoculation needle. After multiple isolation and purification processes at 28℃, a purified isolate, designated XJ-2, was obtained and transferred to PDA slant for growth for 3-4 days, then stored at 4℃. All the above operations were performed in a clean bench.

[0032] 1.1 Verification of Koch's Law:

[0033] The XJ-2 strain was inoculated into corn borer larvae using a spray tower. After inoculation, the larvae died and were cultured for several days in moist, sterile petri dishes (culture conditions: temperature 28℃, culture time approximately 1-2 days). XJ-2 bacteria then grew from the intersegmental membranes of the head, thorax, and abdomen. The bacteria initially turned from bright white to dark yellow, and after isolation and purification, formed pale yellow to brown colonies on PDA medium. When healthy corn borer larvae were inoculated with the bacteria cultured on PDA medium, a large number of larvae died. After several days of moist culture, similar bacteria grew on the dead larvae. These bacteria were then isolated, purified, and cultured on PDA agar, again forming pale yellow to brown colonies. These results indicate that the pathogenicity of XJ-2 to corn borers conforms to Koch's postulates.

[0034] 1.2 Morphological identification of isolated strains:

[0035] The isolated strain XJ-2 initially appeared as white, fluffy colonies on PDA plates, growing in an expanding ring pattern. After 15 days of cultivation on PDA medium, the colony diameter reached 50.53 mm. Conidia were produced after approximately 7 to 10 days of culture, turning the colony yellowish-green or olive-colored due to spore production. The center of the colony was slightly convex, later turning dark brown or chestnut-colored, with a colorless reverse side. Under a microscope, conidiophores and conidia were visible. The conidia were colorless, smooth, and spherical with a few oval shapes. The conidiophores branched forward from the lateral ends of the hyphae, forming new conidia at the apex. Based on these morphological observations, it was preliminarily identified as belonging to the genus *Aspergillus*.

[0036] Morphological observation of strain XJ-2 as follows Figure 2 As shown.

[0037] 1.3 Molecular identification of isolated strains:

[0038] PCR amplification of strain XJ-2 DNA was performed using primers CMD5 (5'-CCGAGTACAAGGAGGCCTTC-3') and CMD6 (5'-CCGATAGAGGTCATAACGTGG-3'). The amplified PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the sequenced results are shown in SEQ ID No. 1. Homology BLASTE alignment of the sequencing results with gene sequences stored in the GanBank database showed that the PCR amplified sequence of strain XJ-2 had over 98% homology with the Aspergillus oryzae sequence. A phylogenetic tree was constructed using MEGA 10.0 software, see [link to MEGA 10.0 software]. Figure 1 .according to Figure 1 The phylogenetic tree shows that strain XJ-2 is aggregated with Aspergillus.

[0039] The preservation information for strain XJ-2 is as follows:

[0040] The deposit name is: Aspergillus tamarii XJ-2, depositary institution: China Center for Type Culture Collection, depositary address: Wuhan University, Wuhan, China; deposit date: April 28, 2023, deposit number: CCTCC NO: M2023661.

[0041] 1.4 Insecticidal range of Aspergillus tamarii XJ-2:

[0042] 1.4.1 Test insect source

[0043] Potato beetle (mixed larval stages), peach fruit moth (mixed larval stages), truncated spider mite (mixed larval stages), tomato leafminer (mixed larval stages). Rearing conditions: temperature (28±1)℃, humidity (80±5)%, 12L:12D light incubator.

[0044] 1.4.2 Preparation of spore suspension

[0045] The purified isolate, *Aspergillus tamarii* XJ-2, was propagated using PDA medium. The isolate was cultured on PDA medium at 26°C for 7–10 days, and conidial powder was collected. This powder was then diluted with 0.1% Tween-80 sterile water to prepare a 1.2 × 10⁻⁶ spore volume. 8 A spore suspension at spores / ml was used for determining the insecticidal range.

[0046] 1.4.3 Inoculation Treatment

[0047] The insecticidal range of strain XJ-2 was tested using potato beetle (mixed larval stages), peach fruit moth (mixed larval stages), truncated spider mite (mixed larval stages), and tomato leafminer (mixed larval stages).

[0048] Each of the above insect species consists of 20 individuals as one replicate, with 3 replicates, i.e., 1.2 × 10⁻⁶ individuals. 8 Sixty insects of one species (mixed larval stages) were treated with a spore suspension at a concentration of spores / ml. The treatment group used a spray tower to spray 1.2 × 10⁻⁶ spores / ml. 8A spore suspension of 10 mL / 20 insects was evenly sprayed onto the surface of the test insects. The dosage was the same as described below. Potato beetles (mixed larval stages), peach fruit moths (mixed larval stages), spider mites (mixed larval stages), and tomato leafminers (mixed larval stages) were treated sequentially, ensuring each insect was covered with the spore suspension. After the solution had evaporated, the insects were placed in rearing boxes and kept at room temperature. The control group used a sterile aqueous solution of 0.1% Tween-80, at a dosage of 10 mL / 20 insects (the dosage is the same as described below). The average cumulative mortality rate was calculated after 7 days. As shown in Table 1, the average cumulative mortality rates after 7 days were 56.33%, 78.45%, 87.33%, and 90.23%, respectively.

[0049] Table 1. Insecticidal range of Aspergillus XJ-2

[0050]

[0051] Four days after inoculation through the spray tower wall, dissection revealed no mycelium in healthy control insects, but a large number of XJ-2 strain mycelium were observed in pathogenic insects. The same substance (XJ-2 strain mycelium) also appeared on the dead insects after moist culturing (at 28℃ for approximately 1-2 days). This demonstrates that *Aspergillus tamarii* XJ-2 kills potato beetles, peach fruit moths, spider mites, and tomato leafminers through body wall infection.

[0052] In summary, the indoor insecticidal range experiment found that Aspergillus tamarii XJ-2 had an indoor mortality rate of over 85% against both the truncated spider mite and the tomato leafminer.

[0053] This shows that Aspergillus has high insecticidal activity against both the truncated spider mite and the tomato leafminer, and it has great potential for biological control of pests.

[0054] 1.5 Indoor toxicity test of Aspergillus XJ-2 against Tetranychus truncatula

[0055] 1.5.1 Test insect source

[0056] Tetranychus truncatula was bred in a 12L:12D light incubator at (28±1)℃ and (80±5)% humidity using common bean (Phaseolus vulgaris) as the host plant. To obtain larvae, nymphs, and adult females of uniform age, this invention involves transferring large numbers of adult females in their peak oviposition period onto prepared common bean leaf dishes. After allowing them to lay eggs for 12 hours, all adult females are removed (resulting in eggs of uniform age). These eggs are then cultured in a light incubator for 4 days until most hatch into larvae (<12 hours). All unhatched eggs are then removed, yielding larvae of uniform age. The resulting larvae can then be used for bioassays. A similar process can be used to obtain the nymphs and adult females to be tested.

[0057] 1.5.2 Preparation of spore suspension

[0058] The purified isolate, *Aspergillus tamarii* XJ-2, was propagated using PDA medium. The isolate was cultured on PDA medium at 26°C for 7–10 days, and conidial powder was collected. This powder was then diluted with 0.1% Tween-80 sterile water to prepare a 1.2 × 10⁻⁶ spore volume. 8 2.1×10 7 3.4×10 6 1.42×10 5 and 1.31×10 4 A spore suspension at spores / ml is used for toxicity assays.

[0059] 1.5.3 Inoculation Treatment

[0060] The leaf spray method was used. A petri dish with a diameter of 9.5 cm and a height of 1.0 cm was selected. A sponge was cut into a circle with a diameter of 9.0 cm and placed in the dish. Water was added until saturated, then filter paper was placed on top. Fresh bean leaves were cut and placed in the dish with the underside facing up, ensuring the leaves were in close contact with the filter paper. The edges and petioles of the leaves were wrapped with absorbent cotton. Each leaf was inoculated with 20 mites (observed under a stereomicroscope, and dead or inactive individuals were removed to ensure 20 mites per leaf). The dish was placed in an insect rearing chamber for 30–60 minutes. The test bacterial suspension was prepared at five concentration gradients (1.2 × 10⁻⁶). 8 2.1×10 7 3.4×10 6 1.42×10 5 and 1.31×10 4Spores / ml), with 0.1% Tween-80 sterile water as a negative control. The above spore suspensions of different concentrations were sprayed onto the spider mites using a spray tower (10 mL). Each treatment had four replicates (four leaves), for a total of 80 spider mites. The control group was sprayed with a solution containing 0.1% Tween-80. After spraying, the petri dishes were placed in an incubator with a humidity of 70% ± 5%, a temperature of (25 ± 2)℃, and a photoperiod of 16L:8D. The number of dead spider mites was observed and recorded daily, and dead mites were removed. The dead mites were placed in petri dishes for moist culture, and the presence of Aspergillus flavus was observed on the mites to determine whether death was caused by Aspergillus flavus infection. Simultaneously, the diseased spider mites that had died from Aspergillus flavus infection were photographed using an optical microscope (Nikon Eclipse80i). The experiment was observed continuously for 7 days. Using the obtained data, the corrected mortality rate of spider mites was calculated using Microsoft Excel 2010, and the lethal median concentration (LC50) and lethal median time (LT50) were calculated using GraphPad Prism 8. The developmental period of the truncated spider mite and the lifespan of female adult mites were also calculated.

[0061] The inoculation treatment for larvae, nymphs, and adult female mites is the same as in step 1.5.3 above.

[0062] Corrected mortality rate (%) = ((treatment mortality rate - control mortality rate)) / ((1 - control mortality rate) × 100%);

[0063] 1.5.4 Indoor toxicity of Aspergillus XJ-2 to larvae and its effects on the developmental period and lifespan of adult female spider mites:

[0064] The test insects used were larvae, and the other steps were the same as in step 1.5.3 above. The results are shown in Tables 2 to 4.

[0065] 1.5.5 Indoor toxicity of Aspergillus XJ-2 to nymphs and its effects on the developmental period and lifespan of adult female spider mites:

[0066] The test insects used were nymphs, and the other steps were the same as in step 1.5.3 above. The results are shown in Tables 2 to 4.

[0067] 1.5.6 Indoor toxicity of Aspergillus XJ-2 to female adult mites and its effects on the developmental period and lifespan of female adult mites of Tetranychus truncatula:

[0068] The test insects used were adult female mites, and other procedures were the same as in implementation 1.5.3. The results are shown in Tables 2 to 4.

[0069] Table 2 Mortality rates of different life stages of *Tetranychus truncatus* under different inoculation concentrations of *Aspergillus oryzae* XJ-2.

[0070]

[0071] Table 3. Changes in LT50 of different life stages of *Tetranychus truncatula* treated with different concentrations of *Aspergillus xJ-2*.

[0072]

[0073] Table 2 presents the results of ANOVA and Turkey's mean test for the mortality rates of larvae, nymphs, and adult females at different concentrations of *Aspergillus oryzae* XJ-2. The mortality rates of the control groups for each mite developmental stage remained at the natural mortality rate (<10%). After 7 days of treatment with different concentrations of bacterial suspension, significant differences in the mortality rates of *Tetranychus truncatula* were observed among different mite developmental stages at different concentrations. Generally, within the same mite developmental stage, the mortality rate of *Tetranychus truncatula* gradually increased with increasing treatment concentration, with the highest mortality rate observed at a spore suspension concentration of 1.2 × 10⁻⁶. 8 Seven days after inoculation with spores / mL, the mortality rates of larvae, nymphs, and adult females were 89.33±1.72%, 93.22±2.31%, and 94.23±1.26%, respectively. At the same concentration, the mortality rate increased with increasing mite age, showing the order of larvae < nymphs < adult females. Adult females were the most susceptible to Aspergillus XJ-2 infection and had the highest mortality rate. This demonstrates that Aspergillus XJ-2 has a high lethal effect on Tetranychus truncatula.

[0074] Table 3 shows that the LT50 of *Tetranychus truncatula* decreased significantly with increasing concentration of *Aspergillus oryzae* XJ-2 suspension. This indicates that *Aspergillus oryzae* XJ-2 has a significant lethal effect on *Tetranychus truncatula*.

[0075] Table 4. Effects of Aspergillus XJ-2 treatment on the developmental period and lifespan of adult females of Tetranychus truncatum.

[0076] strain egg Larvae nymphal mites Era Time Lifespan of female adult mites Aspergillus XJ-2 4.53±0.04a 1.83±0.21a 3.63±0.61a 9.79±0.44a 27.87±2.33a CK 3.91±0.06b 1.71±0.33b 3.01±0.56b 8.63±0.63b 30.23±3.01b

[0077] Table 4 shows that, compared with the control, the developmental period of *Tetranychus truncatula* treated with *Aspergillus XJ-2* was significantly prolonged in the egg, larval, and nymphal stages, and the lifespan of adult female mites in the treatment group was significantly shorter than that in the control group. This indicates that *Aspergillus XJ-2* treatment has a significant impact on the growth and development of *Tetranychus truncatula* offspring.

[0078] 1.6 Evaluation of the greenhouse control efficacy of Aspergillus XJ-2 against Tetranychus truncatula.

[0079] A field trial was conducted on the morning of April 3, 2023, in accordance with the field efficacy test method (Ministry of Agriculture of the People's Republic of China. Guidelines for Field Efficacy Tests of Pesticides (I) Acaricides for the Control of Spider Mites in Legumes and Vegetables: GB / T 17980.17-2000). The pesticide was applied when the larvae and nymphs of the spider mite were just beginning to become active, with an average of more than two live mites per leaf. A Gongnong-16 sprayer was used to evenly spray the bacterial suspension onto both sides of the greenhouse cowpea leaves at a rate of 50 L / mu (approximately 0.067 hectares), ensuring the suspension did not drip. The bacterial suspension was applied only once throughout the entire trial. The number of active mites on 25 leaves was investigated using a handheld magnifying glass. The initial insect population was assessed before application, and again at 3, 5, 7, and 14 days after application. The control effect in each treatment area was calculated. The control efficacy was calculated using the following formula.

[0080] Control efficacy = (1 - (number of mites in control group before treatment × number of mites after treatment / number of mites in control group after treatment × number of mites before treatment) × 100%

[0081] Table 5. Control efficacy of Aspergillus XJ-2 in greenhouse trials.

[0082]

[0083]

[0084] Table 5 shows that 3-14 days after drug administration, the concentration of Aspergillus XJ-2 suspension was 1×10⁻⁶. 8 The spore / mL control efficacy was 34.53%–84.55%, significantly higher than that of medium (1×10⁻⁶). 7 spores / mL and 1×10 6 spores / mL), low (1×10) 5 Treatment with spore / mL bacterial suspension concentration. The control efficacy increased continuously over time, reaching its peak at 14 days post-treatment, ranging from 44.04% to 84.55%.

[0085] 1.7 Indoor toxicity test of Aspergillus XJ-2 against tomato leafminer

[0086] 1.7.1 Indoor bioactivity assay

[0087] The preparation of the spore suspension is the same as in 1.5.2.

[0088] The pathogenicity of Aspergillus XJ-2 against the eggs, larvae of different instars, and pupae of the tomato leafminer was determined using a spray method. Fully unfolded tomato leaves (approximately 8cm at their longest and 5cm at their widest points) were quickly and obliquely cut at the petiole with a sharp utility knife. The leaves were then preserved with absorbent cotton and plastic wrap, and placed in a round plastic insect rearing box (9cm in diameter and 3cm in height) for later use.

[0089] ① Egg bioassay: Tomato leaves bearing pale yellow-green eggs laid by the tomato leafminer were selected. After preservation treatment, the eggs were inoculated with Aspergillus oryzae XJ-2 spore suspension using a spray tower. 30 eggs were treated at each concentration, with 5 replicates. The number of eggs hatching into larvae was observed daily for 7 consecutive days (i.e., until no more larvae hatched or all hatched). Hatching status was recorded, and the egg hatching rate was calculated.

[0090] ② Larval bioassay: A suspension of Aspergillus spores (XJ-2) was inoculated into 1st-2nd, 3rd, and 4th instar larvae using a spray tower. These larvae were then transferred to fresh tomato leaves. Twelve larvae were treated at each concentration and instar, with four replicates. Larval survival was observed every 24 hours for 7 consecutive days. A larvae were considered dead if they showed no reaction and no luster when lightly touched with a soft-bristled brush. Dead larvae were then cultured in petri dishes (D=12cm) under humid conditions for 20 days. The cause of death was determined by the presence and morphology of mycelium. Simultaneously, the larvae infected with Aspergillus spores and subsequently killed were photographed using an optical microscope (Nikon Eclipse80i). The number of dead and surviving larvae was recorded, and the larval mortality rate and corrected mortality rate were calculated. The in vitro toxicity of Aspergillus spores (XJ-2) to different instar larvae of the tomato leafminer was calculated based on the concentration, larval instar, treatment time, and corrected mortality rate.

[0091] ③ Pupal bioassay: Tomato leafminer pupae were inoculated with a suspension of Aspergillus XJ-2 spores using a spray tower, and then placed in petri dishes (D=12cm). Twelve pupae were treated at each concentration and each instar, with four replicates. Pupal emergence was observed every 24 hours for 7 consecutive days (i.e., until emergence ceased or all pupae emerged). Simultaneously, diseased tomato leafminer pupae that had died from Aspergillus infection were photographed using an optical microscope (Nikon Eclipse80i). Pupal emergence was recorded, and the emergence rate was calculated.

[0092] 1.7.2 Effect of Aspergillus XJ-2 on the hatching rate of tomato leafminer eggs

[0093] like Figure 4 As described above, Aspergillus XJ-2 is pathogenic to tomato leafminer eggs. Compared with the control, the hatching rate of eggs inoculated with Aspergillus XJ-2 on day 3 was 80.55%, which was significantly lower than the hatching rate of the control group (100%), indicating that Aspergillus XJ-2 has an adverse effect on egg hatching.

[0094] 1.7.3 Effects of Aspergillus XJ-2 on Corrected Mortality Rate of Tomato Leafminer Larvae

[0095] Aspergillus XJ-2 exhibits insecticidal activity against tomato leafminer larvae, but this activity gradually decreases with increasing larval stage. Ten days after inoculation, the corrected mortality rates of 1st to 4th instar tomato leafminer larvae in the Aspergillus XJ-2 treatment groups were 100%, 82.7%, 81.9%, and 70.1%, respectively. Figure 5 The above indicates that Aspergillus XJ-2 has good insecticidal activity against tomato leafminer larvae.

[0096] 1.7.4 Effect of Aspergillus XJ-2 on the emergence rate of tomato leafminer pupae

[0097] Aspergillus XJ-2 significantly reduced the emergence rate of pupae in the tomato leafminer moth. Eight days after treatment, the emergence rate of pupae in the Aspergillus XJ-2 treatment group was 45.55%, a decrease of 47.67% compared to the control group (93.22%). Figure 7 As described above. Experiments revealed that after 4 days of treatment with *Aspergillus oryzae* XJ-2, some pupae turned black; after 3-5 days of moist cultivation, a small amount of yellowish-green mycelium appeared on the abdominal segments of some pupae. Figure 8 As shown in the figure. These results indicate that strain XJ-2 can effectively infect the pupae of the tomato leafminer moth, leading to a decrease in the emergence rate.

[0098] It should also be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. Aspergillus tamarii XJ-2, characterized by: The accession number is CCTCC NO: M 2023661.

2. The use of Aspergillus XJ-2 as described in claim 1, characterized in that: Used to control truncated spider mites and / or tomato leaf miners.

3. The use of Aspergillus XJ-2 according to claim 2, characterized in that: It can also be used to control potato beetles and / or peach fruit moths.

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